This application relates to amplification of DNA and, in particular, to the simultaneous amplification of multiple target sequences using the Ligase Chain Reaction (hereinafter "LCR"). It is a continuation-in-part of co-owned and co-pending U.S. Ser. No. 07/860,702 filed Mar. 31, 1992, which is incorporated by reference.
This application is related to several other applications relating to LCR, including U.S. Ser. No. 131,936 filed Dec. 11, 1987, now pending, a continuation of that application, U.S. Ser. No. 720,739 filed Jun. 25, 1991, now pending, U.S. Ser. No. 470,674 filed Jan. 26, 1990, now abandoned; and a continuation-in-part from that application, U.S. Ser. No. 634,771 filed Jan. 9, 1991; now pending. It is noted that published EP-A-320 308 corresponds to U.S. Ser. No 131,936, and published EP-A-439 182 corresponds to U.S. Ser. No. 634,771. Both of the published documents in their entirety are incorporated herein by reference.
LCR is a method for amplifying exponentially the number of detectable target molecules. It involves the use of two pairs of probes. A first or primary pair hybridizes with one strand of a target sequence at near-adjacent positions so that they can be ligated together in template-dependent fashion to form a reorganized primary molecule. The secondary pair is capable of hybridizing to the reorganized primary molecule. LCR was first described by Backamn, et al. in EP-A-320 308. Much has been written about it since then. For example, see Wallace, EP-A-336 731; Orgel, WO 89/09835; Richards, WO 89/12696; Segev, WO 90/01069; and Barany, Proc. Natl. Acad. Sci USA 88:189-193 (1991). A variation of LCR known as "Gap" LCR is described in EP-A439 182 and in Segev, WO 90/01069.
Instead of using two pairs of probes capable of forming blunt-ended duplexes, at least one probe of one of the probe pairs initially includes a "modified" end which renders the resultant duplex "nonblunt" and/or not a suitable substrate for the ligase catalyzed fusion of the two probe duplexes. A "modified end" has either (1) a blocking moiety (or additional base residues) on a group (e.g. the 5' phosphate or the 3' hydroxyl) which, under ordinary LCR conditions, obligatorily participates in the ligase catalyzed fusion or (2) omitted bases to create a "gap" between one probe terminus and the next probe terminus In the "gap" embodiment, modified ends are created by eliminating from one or more of the probes a short sequence of bases, thereby leaving a recess or gap between the 5' end of one probe and the 3' end of the other probe when they are both hybridized to the target (or target complement, or polynucleotide generated therefrom). In order for LCR to amplify the target, the gaps between the probes must be filled in (i.e., the modification must be "corrected"). In a first version, this can be done using a polymerase or a reverse transcriptase and an excess of deoxynucleotide triphosphates which are complementary to the target strand opposite the gap. Alternatively, this can be done by supplying a fifth probe complementary to the target and a sixth probe complementary to the fifth probe.
PCR or polymerase chain reaction is a different method for amplifying DNA. It employs two primers which hybridize to opposite strands of a double stranded target. A polymerase initiates extension of the primer using the target as a template by sequentially adding the appropriate complementary nucleotides. PCR is described in U.S. Pat. Nos. 4,883,195 and 4, 883,202, the entire disclosures of which are incorporated herein by reference.
PCR has been used in a multiplex manner to determine the presence of multiple target sequences in a single reaction. EP-A-364 255 describes the use of multiple primer sets to simultaneously amplify multiple target sequences by PCR. A similar disclosure is made in Chamberlain, et al., Nucl. Acids Res., 16:1141-56 (1988).
In addition, Nickerson, et al., Proc. Natl. Acad. Sci. USA, 87:8923-8927 (1990) proposes an oligonucleotide ligation assay ("OLA") for multiple target sequences, pending the development of "multiple, nonisotopic reporter groups". OLA employs two contiguous probes that are ligated together and the ligated product is detected as a measure of the presence of a target sequence.
In spite of the existence of these disclosures, multiplex LCR is not available in the hands of the public. The an does not provide sufficient guidance actually to enable the concept of multiplex LCR. This is due largely to the inapplicability of PCR conditions to LCR.